UNIVERSITI TUNKU ABDUL RAHMAN

FACULTY OF SCIENCE

BACHELOR OF SCIENCE (HONS) CHEMISTRY

YEAR 1 SEMESTER 1

UDEC 1224 CHEMISTRY LABORATORY II

Name : Deevyar a/p S.Lingam

Experiment : Experiment 9

Title : Thin Layer Chromatography (TLC)

Date : 15/6/2016

Date of Submission : 19/6/2016

Time : 3.00 – 5.30 p.m.

Lecturer : Dr.Neo Kian Eang

Practical Group : P7

Group Members : Long Mei Fang 1505485

Angeline Devadason 1403358

1.0 Title

Thin-Layer Chromatography

2.0 Objectives

 To learn the technique of TLC and the visualization of colourless components

 To identify an unknown drug by a TLC comparison with Standard compounds

3.0 Introduction

Chromatography is a common and powerful method used in the separation of two or

more compounds or ions by the distribution between two phases, one which is moving and
the other which is stationary. The principle of works of chromatography is that different
compounds will have different solubilities and adsorption to the two phases between which
they are portioned. The material is carried along by the mobile phase after the separation of
the material that is place onto the stationary phase. The stationary phase absorbed the
components of the mixture to different degrees and it is thus the different speeds of migration
for each component on the adsorption the slower the material passes through the system. The
techniques of thin layer chromatography will be learning in this experiment.

The phase each component remains in and the rate at which each travels depends by
the separation of the components of a mixture. The other mobile phase travels past the fixed
phase while the stationary phase remains still. Each compounds travel past the stationary
phase at a different rate due to various functional groups. The strength of the following types
of interactions: ion-dipole, and van der Waals forces.

Various types of stationary and mobile phases used by different types of

chromatography. In this experiment, the mobile phase is an organic solvent (may be a single
type or a mixture of solvents) and the solid phase is silica gel. This means that the compounds
to be separated must choose between being absorbed to the solid silica gel or moving along in
the organic solvent. The silica gel is adhered to a sheet of glass, plastic or aluminium.

The polarity of the solvent is a factor that establishes the speed at which a compound
travels past silica gel. The compounds are disallow to compete for silica absorption sites as
the polar solvent is competing for the sites. This encourage a faster rate at which all
compounds travel. The sequence in which the compounds travel remains the same, while
travelling faster as the polarity of the eluent ( solvent system) increases.

The same principles of separation as column chromatography is involved for TLC but
the apparatus and technique for development is different. The silica ( or alumina) is adhered
to a plate of glass, plastic or aluminium instead of a column. The dissolved sample are used
to apply by a capillary spotter I onto the plate about one cm from the bottom (aline with
pencil is drawn). Only the sample remains once the solvent has evaporated. A closed
developing chamber was place carefully by the plate , which as a shallow layer of solvent that
does not submerse the spot. A folded piece of filter paper was lined by the chamber to ensure
a uniform and saturated atmosphere of solvent vapour. When the solvent front has reached
about 0.5 cm from the top, the plate is removed and is quickly marked with pencil. The initial
spot to be separated into individual components is cause by the capillary action of the solvent
that may be visualized by colour identification or with the following techniques for colourless
compounds:

a) Irradiation with ultraviolet light

b) Reversible staining with iodine vapour (formation of brown spot which fade)
c) Spraying with a reagent that irreversibly colours the spots, e.g H2SO4, KMNO4.
The rate at which a compound moves in respect to the solvent front, Rf , is characteristics of
that compound under standard conditions. The Rf value is calculated by dividing the distance
each spot has travelled (measures from the pencil line to the middle of the spot) by the
distance the solvent front travelled from the pencil line. The benefits of TLC are the very
small quantities of sample required and the great ease and rapidly with which it is resolved.
For this reasons, it is often used to monitor the progress of a reaction by running the crude
sample beside the reaction sample on the same plate. The following are some common uses
of TLC:

a) To determine the number of components in a mixture

b) To determine the identity of two substances
c) To monitor the progress of a reaction’
d) To determine the effectiveness of a purification
e) To determine the appropriate conditions for a column chromatographic separations
f) To monitor column chromatography
The weight ratio of adsorbent to sample is significant to get the accurate separation. The
active adsorbing sites will be saturated if too much sample is applied resulting in poor
separations. The solvent (or solvent mixture) is significant to the compound separation. The
compound will move faster as the polarity of the solvents increases. In some cases, a solvent
system may increases in polarity by gradually changing the composition of the solvent
mixture.

Cyclohexane

n-hexane (non-polar)

benzene

toluene

dichloromethane increasing eluting

chloroform power with polar

diethyl ether Increasing polarity stationary phases

ethyl acetate

acetone

ethanol

methanol

acetonitrile

water (polar)

Compounds with highly polar groups are strongly adsorbed and eluted less readily than
less polar (or polarizable) compounds. Below are the listed order of the types of polar
functional groups decreases for the strengths of adsorption for compounds.

RH, R-X, alkenes, R-OCH3, R-CO2R, RCOR,RNH2, R-OH, R-CO2H

Increasing absorption on polar stationary phases

4.0 Materials

 Aspirin  TLC plates

 Acetaminophen  Ethyl acetate
 Unknown A  Hexane
 Caffeine  Acetic acid
 Unknown B  Iodine

5.0 Apparatus

 UV lamp
 Capillary tube
 250 mL beaker

6.0 Procedure

1) A TLC plates can be obtained from your instructor.

2) The plate was set down on a clean, dry surface then a line was drawn LIGHTLY across
the plate about one cm from the bottom of the plate with the help of a 2B pencil.
3) Next five 2-3mm lines was make, spaced about 0.8cm apart and was running
perpendicularly through the lines across the bottom of the TLC plate.
4) The plate was spotted with 5 different analgesic. A separate capillary tube was used for
each sample. The acetaminophen was spotted first, then followed by the caffeine, then
the unknown A, then aspirin, and lastly the unknown B and the spots was made as small
as possible.
5) The plate was examined under the ultraviolet(UV) light to see that enough for each
compound has been applied otherwise it will be added more to it.
6) A developing chamber was prepared using a 250mL beaker as a chamber and aluminium
foil to cover.
7) The eluent, 2:1 mixture of ethyl acetate : hexane, was poured into the beaker to a depth
of under one cm( about 15mL). About 5 drops of acetic acid was added into the beaker
and was mixed.
8) The prepared TLC plate was placed in the developing chamber , ensuring the solvent
level was below the pencil line.
9) The plate was removed after the solvent has risen to near the top of the plate( about
0.5cm from the top) and the solvent front was marked with a pencil.
10) The solvent was allowed to be evaporated from the plate in the fumehood.
11) The colourless compounds was visualized by illumination of the plate with an
ultraviolet(UV) lamp. A bright florescence was shown for many substances, particularly
aromatic compounds which may have a characteristics colour.
12) The spots was outlined with a 2B pencil.
13) The plate was sketched in your notebook and the Rf values was calculated for each spot.
14) The unknown drug was determined based on the Rf values.

7.0 Results

A B C D E

acetaminophen caffeine unknown A aspirin unknown B

 Distance moved (cm)

Lanes Substances spotted Rf values

Spot Solvent

A Acetaminophen 3.9 7.8 2.0

B Caffeine 1.8 7.8 0.2

C Unknown A 3.9 7.8 2.0

D Aspirin 6.2 7.8 1.3

E Unknwon B 6.2 7.8 1.3

8.0 Discussion

In the experiment, eluent was used which is a mixture of ethyl acetate : hexane in a
ratio of 2 : 1. Ethyl acetate is a polar solvent because of the oxygen bonded to carbon in the
compounds has higher electronegativity than carbon. This causes a partial negative charge on
the oxygen atom of the ethyl acetate and partial positive charge on the carbon atom.
However, hexane is an organic solvent which is non-polar. Therefore, eluent polarity
properties was given by the mixture of the two solvents and the polar compounds was
allowed to be separated as well. The polar compounds will not be able to carry by the eluent
and will not be separated if the polarity of the eluent is too low. However, the polar
compound will move in a higher rate if the polarity of the eluent is too high. This will causes
the separation between non-polar compound and polar compound to become so small which
will result in poor separation. Hence, during chromatography, the polarity of the eluent must
be optimal for the separation of the samples. A few drops of acetic acid also has been added
to the eluent in order to prevent the deprotonation of the compounds during the separation of
compounds as it is necessary for retaining the identity of the compounds and hence, a pure
compounds can be separated out.
 TLC plate have been used for the separation of the compounds. The TLC plate were
coated by silica gel and act as the adsorbent. The difference in electronegativity of the oxygen
atom with the silicon atom is due to the polar functional group such as Si-O-Si and Si-OH
that was consisted by the silica gel. Polar compounds will then interact strongly with the
silica gel and adsorb on the silica gel strongly since the silica gel is a polar. Therefore, only a
short distance will be travelled by the polar compounds from the bottom of the TLC plates as
the eluent move upward and for the non-polar compounds, they will interact weakly with the
silica gel causing it to be easily carried by the eluent. The non-polar compounds can travel
further away from the bottom of the TLC plate as the eluent moving upwards. The capillary
action of the solvent causes the movement of the eluent along the TLC plate, allowing the
solvent to travel in the gap present in the three dimensional network of silica gel. This causes
the initial spot to be separated into individuals components as the samples were carried by the
eluent. The eluent vapour will remain in the chamber since the developing chamber is
covered with aluminium foil. The eluent will move upwards quicker along the silica gel
without vaporization when the chamber is saturated with eluent vapour which eventually
promotes the rate of eluent travelled along the silica gel without affecting the separation of
the compounds.

Acetaminophen, caffeine and aspirin compounds were spotted on the TLC plate. Below are
the diagrams that show the structure of this compounds;

acetaminophen caffeine aspirin

All of the three compounds above are polar compounds but the polarities of the compounds
may differ from each other due to the electronegativity difference between the atoms in the
compound. Carbon atom and hydrogen atom present in these compounds have lesser
electronegativity compared to the nitrogen and oxygen atom. This causes a partial positive
charge for the carbon and hydrogen atoms and a partial negative charge for nitrogen and
oxygen atoms.

Based on the results obtained, aspirin travelled further away than acetaminophen and
caffeine. This can be clearly shown by the calculation of the Rf values of each compound
whereby the higher Rf value indicate that the compound can travel further away and thus is
less polar. The Rf value for aspirin is 1.3, followed by acetaminophen which is 2.0 and finally
the caffeine which is 0.2. Therefore, the polarity of the solvent increases from aspirin to
acetaminophen and finally to caffeine. This is due to the presence of large number of
electronegativity atom such as nitrogen and oxygen atom in the compounds, thus the polarity
of the caffeine compound increases. We can identify the unknown compounds since the
compounds with similar polarities will travel with the same distance along the TLC plate by
comparing the Rf values of the unknowns with the standard compounds. Unknown A and
unknown B have the Rf values of 2.0 and 1.3 respectively. Since unknown A has the same Rf
value as acetaminophen, thus we can conclude that unknown A is acetaminophen whereas for
unknown B, it has the same Rf value as aspirin, so we can deduce that unknown B belongs to
the aspirin.

There are some of the precaution steps that should be considered during the experiment
to avoid problems. First of all, safety goggle should be wore throughout the experiment to
prevent chemicals from entering the eye. Secondly, when handling the UV light, do not look
directly at the ultraviolet light as it can cause eye injuries. Thirdly, do not interrupt the beaker
after TLC plate has been introduced into the developing chamber as it can cause error to the
measurement. Finally, do not bend the silica gel excessively since the silica adsorbent may
flake off.

9.0 Conclusion

From this experiment, we are able to study the technique of using the TLC method
whereby the thin layer chromatography (TLC) is a method for analysing mixtures by
separating the compounds in the mixture. TLC helps in determining the number of
components in a mixture, the identity of compounds, the purify of a compound and also the
progress of a reaction. We are also able to visualized the colourless components on the TLC
plate under an ultraviolet light(UV) which shows different colour for every each components.
On the other hand, an unknown drug was able to be identified by a TLC comparison with
Standard compounds. The comparison of the Rf values with every each of the compounds
determine the type of the unknown drug. The Rf value for acetaminophen and unknown A is
the same which is 2.0 whereas the Rf value for aspirin and unknown B is the same which is
1.3. The Rf value for caffeine is not the same as for unknown A and unknown B which is 0.2.
From the calculation, it is clearly shown that unknown A is acetaminophen and unknown B is
aspirin.
10.0 References

Chemguide; Clark. J. 2007. Paper Chromatography. [ONLINE] Available at:

http://www.chemguide.co.uk/analysis/chromatography/paper.html. [Accessed 19 June 2016].

Jeffrey. 2011. One of Chemistry. [ONLINE] Available at:

http://oneofchemistry.blogspot.my/2011/10/thin-layer-chromatography-and-column.html.
[Accessed 18 June 2016].

Mendelset ; Matt. 2011. Thin Layer Chromatography (TLC). [ONLINE] Available at:
http://www.mendelset.com/articles/683/thin_layer_chromatography_tlc. [Accessed 18 June
2016].

Odinity ; Mickey, L. 2014. Thin Layer Chromatography and Column Chromatography

Experiment. [ONLINE] Available at: http://www.odinity.com/thin-layer-chromatography/.
[Accessed 20 June 2016].

Blogspot ; Writer, B. 2011. One Part of Chemistry. [ONLINE] Available at:

http://1chemistry.blogspot.my/2011/11/thin-layer-chromatography-and-column.html.
[Accessed 18 June 2016].

Blogspot ; ynia, y. 2013. lab report:THIN LAYER CHROMATOGRAPHY OF

ANALGESICS DRUGS. [ONLINE] Available
at:http://yunayunia.blogspot.my/2013/10/thin-layer-chromotography-of-analgesic.html.
[Accessed 20 June 2016].